The leaves of Stevia rebaudiana of the Asteraceae family contain a secondary metabolite called “steviol” which is a kind of diterpenoid. Steviol glycosides, which are products of the addition of sugars to steviol, include those having sweetness about 300 times higher than that of table sugar. Such steviol glycosides are used as non-caloric sweeteners in the food industry.
Obesity is becoming more of a serious social issue on an international scale, and non-caloric sweeteners are increasingly demanded from the viewpoint of promotion of health and reduction of medical cost. Currently, aspartame and acesulfame potassium, which are artificially-synthesized amino acid derivatives, are used as artificial sweeteners. However, naturally-occurring non-caloric sweeteners such as steviol glycosides are expected to be safer and gain more public acceptance.
Among steviol glycosides, stevioside is a compound in which three glucose units are added to steviol, and is contained in the largest amount in the leaves of common Stevia rebaudiana. Stevioside has a degree of sweetness about 300 times higher than that of sucrose, but has slightly bitter taste. Rebaudioside A, which is another steviol glycoside, is a compound in which four glucose units are added to steviol, and has a degree of sweetness about 400 times higher than that of sucrose. Stevioside and rebaudioside A are primary substances responsible for the sweetness of Stevia rebaudiana. There are also known glycosides such as rebaudioside D in which five glucose units are added to steviol and rebaudioside M in which six glucose units are added to steviol. It is also known that Rubus suavissimus contains rubusoside in which one glucose unit is added at each of the 13 and 19 positions of steviol and that this rubusoside is a primary sweet component of Rubus suavissimus. In addition to the above glycosides, glycosides considered to be reaction intermediates and analogs differing in the type of sugar are known to exist (FIG. 1).
Meanwhile, steviol is known to have, for example, improving effect on cognitive function.
If an enzyme acting only on a specific glycoside bond in steviol glycosides can be used, production of a specific glycoside or elimination of an unnecessary glycoside will become possible. This will bring a lot of merits such as facilitating the improvement in taste of Stevia rebaudiana extracts or the purification of a specific steviol glycoside.
An enzyme activity to hydrolyze steviol glycosides has been reported to be observed in some organism species. In particular, concerning the production of steviol glycoside-hydrolyzing enzymes by filamentous fungi of the genus Aspergillus, it has been reported that raw soy sauce has an activity to hydrolyze stevioside into rubusoside (Non Patent Literature 1) and that a pectinase enzyme agent, hesperidinase enzyme agent, and takadiastase enzyme agent have an activity to hydrolyze stevioside into steviol (Non Patent Literatures 2 to 4). A method has also been reported in which steviol is produced from stevioside by the combined use of a pectinase enzyme agent derived from filamentous fungi of the genus Aspergillus and an enzyme agent derived from Helix pomatia (Patent Literature 1). Viscozyme L (novozyme), an enzyme agent derived from Aspergillus aculeatus, has been described to have an activity to hydrolyze stevioside into rubusoside and then into steviol monglycosyl ester (Non Patent Literature 5). Additionally, an extract obtained from Aspergillus aculeatus by solid culture has been described to have an activity to convert stevioside into steviol (Non Patent Literature 6).
As stated above, filamentous fungi of the genus Aspergillus, including koji mold, have been suggested to have an enzyme gene having steviol glycoside-hydrolyzing activity. However, there has been no report of any gene or enzyme responsible for enzyme activity.
It has been reported that the β-glucosidase of the glycoside hydrolase (GH) family 3 encoded by the AO090009000356 gene of koji mold hydrolyzes disaccharides with a β-glucoside bond (Non Patent Literature 7). Specifically, its specificity for hydrolysis is the highest for laminaribiose with a β-1,3 linkage, followed by β-gentiobiose with a β-1,6 linkage, cellobiose with a β-1,4 linkage, and sophorose with a β-1,2 linkage. However, there has been no report on whether the β-glucosidase has an activity to hydrolyze terpene glycosides typified by steviol glycosides.
Some other organisms have also been reported to have an activity to hydrolyze steviol glycosides. For example, it has been disclosed that bacteria of the genus Clavibacter have an enzyme that decomposes the glucosyl ester bond at the 19 position of rubusoside but does not decompose the glucoside bond at the 13 position (Patent Literature 2). Additionally, it has been reported that Flavobacterium johnsoniae-derived β-glucosidase has an activity to decompose steviol glycosides (an activity to hydrolyze the β-glucoside bond at the 13 position and the glucosyl ester bond at the 19 position).
Although these have been found to have an activity to hydrolyze steviol glycosides, the gene responsible for this activity has not been identified.
Moreover, koji mold contains a large number of genes considered to encode GH3 family or GH5 family enzymes having β-glucosidase-like activity, and thus, even if an enzyme activity can be detected, it is not easy to determine which gene is responsible for the activity.